Journal of General Microbiology (1 987), 133, 3299-33 12. Printed in Great Britain 3299 Genetical and Molecular Studies on gevM, a New Developmental LOCUS of Bacillus subtilis By RACHEL L. SAMMONS,* GILLIAN M. SLY" AND DEREK A. SMITH Department of Genetics, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK (Received 4 June 1987; revised 3 August 1987) A transposon Tn917 insertion between gerE and ilvB has identified a new developmental locus, gerM, in Bacillus subtilis. gerM96 : : Tn917 affects both sporulation and germination. DNA on either side of the transposon has been cloned and includes the previously cloned sdhC and gerE loci. gerE terminates 2.1 kb from the end of the transposon. The gerM96::Tn917 mutant is oligosporogenous,yielding approximately I %of the number of wild-type heat resistant spores in liquid medium and 10% on solid medium. Six hours after the onset of sporulation alkaline phosphatase and glucose dehydrogenase levels were 90% and 7%, respectively, of those of the wild-type. At this time 50% of the mutant cells were still dividing. The occurrence of multiple polar septa and 'pygmy' cells suggested a block at stage I1 of sporulation. Following addition of germinants, mutant spores prepared on nutrient agar lost heat resistance normally but released slightly less dipicolinic acid than wild-type spores. They also showed only partial loss of optical density, associated with a phase-grey appearance and striations in the cortex suggesting partial degradation. Expression of the gerM gene was monitored by production of Q-galactosidase encoded by a promotorless lac2 gene fused to the gerM96 : : Tn917 insertion. It occurred 1 - 5 4h after commencement of sporulation. Transcription was directed from a promoter on the gerE side of gerM and was unaffected by a mutation in the gerE gene. INTRODUCTION Germination in bacteria involves the conversion of a dormant spore into a metabolically active cell. In Bacillus subtilis this process requires the expression during sporulation of many germination (ger) and sporulation (spo) genes to equip the spore with the components necessary for germination (Piggot & Coote, 1976; Losick & Youngman, 1984). Although over 50 loci at which spo or ger mutations arise are known it is likely that more remain to be discovered (Losick & Youngman, 1984). Few of the products of spo and ger genes are known but patterns of their order and interdependence of expression are emerging (Errington & Mandelstam, 1986a, b; Turner et al., 1986). During sporulation some genes are expressed in the mother-cell compartment and others in the forespore (de Lencastre & Piggott, 1979; Errington & Mandelstam, 19866; Turner et al., 1986). It is also likely that some spo and ger genes serve not only to form a particular spore component but to stimulate the expression of other genes, and mutations in such genes result in the pleiotropy which is characteristic of many spo and ger mutants. In this paper we describe the use of transposon Tn917 to identify a new ger locus. The advantages of using this method are firstly that Tn917 confers resistance to macrolide, lincosamide and streptogramin B type antibiotics (MLSR), a character which is readily selectable and thus permits indirect selection of the associated Ger mutant phenotype. Secondly, DNA on either side of the insertion is easily cloned (Youngman et al., 1984). Thirdly, lac2 fusions can be created which may permit the expression of the gene in which the insertion has Abbreviations: DPA, dipicolinic acid ; MUG, methylumbelliferyl p-Dgalactoside. 0001-4215 0 1987 SGM